A Pentavalent Shigella flexneri LPS-Based Vaccine Candidate Is Safe and Immunogenic in Animal Models.

A multivalent vaccine is much needed to achieve protection against predominant Shigella serotypes. Recently, we demonstrated the clinical applicability and immunogenic potential of tri-acylated S. flexneri 2a lipopolysaccharide (Ac3-S-LPS). Using a similar approach, we designed a pentavalent LPS candidate vaccine against S. flexneri 1b, 2a, 3a, 6, and Y (PLVF). In this study, we performed molecular and antigenic characterization of the vaccine candidate and its preclinical evaluation. There were no signs of acute toxicity after subcutaneous administration of PLVF in rabbits at a proposed human dose of 125 µg. No pyrogenic reactions and adverse effects associated with chronic toxicity after repeated administration of PLVF were revealed either. The immunization of mice with PLVF led to ≥16-fold increase in S. flexneri 1b-, 2a-, 3a-, 6-, and Y-specific antibodies. In a serum bactericidal antibody (SBA) assay, we registered 54%, 66%, 35%, 60%, and 60% killing of S. flexneri 1b, 2a, 3a, 6, and Y, respectively. In the guinea pig keratoconjunctivitis model, the efficacy was 50% to 75% against challenge with all five S. flexneri serotypes. These studies demonstrate that PLVF is safe, immunogenic over a wide range of doses, and provides protection against challenge with homologous S. flexneri strains, thus confirming the validity of pentavalent design of the combined vaccine.

Molecular Lipopolysaccharide Di-Vaccine Protects from Shiga-Toxin Producing Epidemic Strains of Escherichia coli O157:H7 and O104:H4.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) O157:H7 and O104:H4 strains are important causative agents of food-borne diseases such as hemorrhagic colitis and hemolytic-uremic syndrome, which is the leading cause of kidney failure and death in children under 5 years as well as in the elderly. METHODS: the native E. coli O157:H7 and O104:H4 lipopolysaccharides (LPS) were partially deacylated under alkaline conditions to obtain apyrogenic S-LPS with domination of tri-acylated lipid A species-Ac3-S-LPS. RESULTS: intraperitoneal immunization of BALB/c mice with Ac3-S-LPS antigens from E. coli O157:H7 and O104:H4 or combination thereof (di-vaccine) at single doses ranging from 25 to 250 µg induced high titers of serum O-specific IgG (mainly IgG1), protected animals against intraperitoneal challenge with lethal doses of homologous STEC strains (60-100% survival rate) and reduced the E. coli O157:H7 and O104:H4 intestinal colonization under an in vivo murine model (6-8-fold for monovalent Ac3-S-LPS and 10-fold for di-vaccine). CONCLUSIONS: Di-vaccine induced both systemic and intestinal anti-colonization immunity in mice simultaneously against two highly virulent human STEC strains. The possibility of creating a multivalent STEC vaccine based on safe Ac3-S-LPS seems to be especially promising due to a vast serotype diversity of pathogenic E. coli.

The combined action of CTCF and its testis-specific paralog BORIS is essential for spermatogenesis.

CTCF is a key organizer of the 3D genome. Its specialized paralog, BORIS, heterodimerizes with CTCF but is expressed only in male germ cells and in cancer states. Unexpectedly, BORIS-null mice have only minimal germ cell defects. To understand the CTCF-BORIS relationship, mouse models with varied CTCF and BORIS levels were generated. Whereas Ctcf+/+Boris+/+, Ctcf+/-Boris+/+, and Ctcf+/+Boris-/- males are fertile, Ctcf+/-Boris-/- (Compound Mutant; CM) males are sterile. Testes with combined depletion of both CTCF and BORIS show reduced size, defective meiotic recombination, increased apoptosis, and malformed spermatozoa. Although CM germ cells exhibit only 25% of CTCF WT expression, chromatin binding of CTCF is preferentially lost from CTCF-BORIS heterodimeric sites. Furthermore, CM testes lose the expression of a large number of spermatogenesis genes and gain the expression of developmentally inappropriate genes that are "toxic" to fertility. Thus, a combined action of CTCF and BORIS is required to both repress pre-meiotic genes and activate post-meiotic genes for a complete spermatogenesis program.

Hypomorphic mTOR Downregulates CDK6 and Delays Thymic Pre-T LBL Tumorigenesis.

PI3K/AKT/mTOR pathway hyperactivation is frequent in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL). To model inhibition of mTOR, pre-T-cell lymphoblastic leukemia/lymphoma (pre-T LBL) tumor development was monitored in mice with T lymphocyte-specific, constitutively active AKT (Lck-MyrAkt2) that were either crossed to mTOR knockdown (KD) mice or treated with the mTOR inhibitor everolimus. Lck-MyrAkt2;mTOR KD mice lived significantly longer than Lck-MyrAkt2;mTOR wild-type (WT) mice, although both groups ultimately developed thymic pre-T LBL. An increase in survival was also observed when Lck-MyrAkt2;mTOR WT mice were treated for 8 weeks with everolimus. The transcriptional profiles of WT and KD thymic lymphomas were compared, and Ingenuity Pathway Upstream Regulator Analysis of differentially expressed genes in tumors from mTOR WT versus KD mice identified let-7 and miR-21 as potential regulatory genes. mTOR KD mice had higher levels of let-7a and miR-21 than mTOR WT mice, and rapamycin induced their expression in mTOR WT cells. CDK6 was one of the most downregulated targets of both let-7 and miR21 in mTOR KD tumors. CDK6 overexpression and decreased expression of let-7 in mTOR KD cells rescued a G1 arrest phenotype. Combined mTOR (rapamycin) and CDK4/6 (palbociclib) inhibition decreased tumor size and proliferation in tumor flank transplants, increased survival in an intravenous transplant model of disseminated leukemia compared with single agent treatment, and cooperatively decreased cell viability in human T-ALL/LBL cell lines. Thus, mTOR KD mice provide a model to explore drug combinations synergizing with mTOR inhibitors and can be used to identify downstream targets of inhibition.

Insights into the Mechanism of Action of Highly Diluted Biologics.

The therapeutic use of Abs in cancer, autoimmunity, transplantation, and other fields is among the major biopharmaceutical advances of the 20th century. Broader use of Ab-based drugs is constrained because of their high production costs and frequent side effects. One promising approach to overcome these limitations is the use of highly diluted Abs, which are produced by gradual reduction of an Ab concentration to an extremely low level. This technology was used to create a group of drugs for the treatment of various diseases, depending on the specificity of the used Abs. Highly diluted Abs to IFN-γ (hd-anti-IFN-γ) have been demonstrated to be efficacious against influenza and other respiratory infections in a variety of preclinical and clinical studies. In the current study, we provide evidence for a possible mechanism of action of hd-anti-IFN-γ. Using high-resolution solution nuclear magnetic resonance spectroscopy, we show that the drug induced conformational changes in the IFN-γ molecule. Chemical shift changes occurred in the amino acids located primarily at the dimer interface and at the C-terminal region of IFN-γ. These molecular changes could be crucial for the function of the protein, as evidenced by an observed hd-anti-IFN-γ-induced increase in the specific binding of IFN-γ to its receptor in U937 cells, enhanced induced production of IFN-γ in human PBMC culture, and increased survival of influenza A-infected mice.

CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention.

The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. Here, we demonstrate that a 79-aa region within the CTCF N terminus is essential for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N terminus of CTCF fused to artificial zinc fingers was not sufficient to redirect cohesin to non-CTCF binding sites, indicating a lack of an autonomously functioning domain in CTCF responsible for cohesin positioning. BORIS (CTCFL), a germline-specific paralog of CTCF, was unable to anchor cohesin to CTCF DNA binding sites. Furthermore, CTCF-BORIS chimeric constructs provided evidence that, besides the N terminus of CTCF, the first two CTCF zinc fingers, and likely the 3D geometry of CTCF-DNA complexes, are also involved in cohesin retention. Based on this knowledge, we were able to convert BORIS into CTCF with respect to cohesin positioning, thus providing additional molecular details of the ability of CTCF to retain cohesin. Taken together, our data provide insight into the process by which DNA-bound CTCF constrains cohesin movement to shape spatiotemporal genome organization.

Transcription factors IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses.

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells.

Highly homogenous tri-acylated S-LPS acts as a novel clinically applicable vaccine against Shigella flexneri 2a infection.

Shigellosis, a major cause of diarrhea worldwide, exhibits high morbidity and mortality in children. Specificity of Shigella immunity is determined by the structure of the main protective O-antigen polysaccharide component incorporated into the lipopolysaccharide (LPS) molecule. Endotoxicity, however, precludes LPS clinical use. Thus, there is still no vaccine against the most prevalent shigellosis species (serotype S. flexneri 2a), despite ongoing efforts focused on inducing serotype-specific immunity. As LPS is highly heterogenous, we hypothesized that more homogenous pools of LPS might be less toxic. We developed a method to generate a homogenous S. flexneri 2a LPS subfraction, Ac3-S-LPS, containing long chain O-specific polysaccharide (S-LPS) and mainly tri-acylated lipid A, with no penta- and hexa-acylated, and rare tetra-acylated lipid A. Ac3-S-LPS had dramatically reduced pyrogenicity and protected guinea pigs from shigellosis. In volunteers, 50 µg of injected Ac3-S-LPS vaccine was safe, with low pyrogenicity, no severe and few minor adverse events, and did not induce pro-inflammatory cytokines. In spite of the profound lipid A modification, the vaccine induced a prevalence of IgG and IgA antibodies. Thus, we have developed the first safe immunogenic LPS-based vaccine candidate for human administration. Homogenous underacetylated LPSs may also be useful for treating other LPS-driven human diseases. Clinical trial registry: http://grls.rosminzdrav.ru/.

3'Igh enhancers hs3b/hs4 are dispensable for Myc deregulation in mouse plasmacytomas with T(12;15) translocations.

Myc-deregulating T(12;15) chromosomal translocations are the hallmark cytogenetic abnormalities of murine plasmacytomas (PCTs). In most PCTs, the immunoglobulin heavy chain (Igh) locus is broken between the Eµ enhancer and the 3' regulatory region (3'RR), making the latter the major candidate for orchestrating Myc deregulation. To elucidate the role of the Igh3'RR in tumorigenesis, we induced PCTs in Bcl-xL-transgenic mice deficient for the major Igh3'RR enhancer elements, hs3b and hs4 (hs3b-4-/-). Contrary to previous observations using a mouse lymphoma model, which showed no tumors with peripheral B-cell phenotype in hs3b-4-/- mice, these animals developed T(12;15)-positive PCTs, although with a lower incidence than hs3b-4+/+ (wild-type, WT) controls. In heterozygous hs3b-4+/- mice there was no allelic bias in targeting Igh for T(12;15). Molecular analyses of Igh/Myc junctions revealed dominance of Sµ region breakpoints versus the prevalence of Sγ or Sα in WT controls. Myc expression and Ig secretion in hs3b-4-/- PCTs did not differ from WT controls. We also evaluated the effect of a complete Igh3'RR deletion on Myc expression in the context of an established Igh/Myc translocation in ARS/Igh11-transgenic PCT cell lines. Cre-mediated deletion of the Igh3'RR resulted in gradual reduction of Myc expression, loss of proliferative activity and increased cell death, confirming the necessity of the Igh3'RR for Myc deregulation by T(12;15).

Transcriptional activation of p21Waf1 contributes to suppression of HR by p53 in response to replication arrest induced by camptothecin.

The inhibitory effect of p53 on homologous recombination (HR) is exerted through sequestration of replication protein A (RPA). Release of the p53/RPA complex in response to replication stress is crucially dependent on the phosphorylation status of both proteins and is required for efficient DNA repair by HR. Phosphorylation of RPA within its RPA2 subunit by cyclin-dependent kinases (CDK) is an early event in the replication stress response. Here we investigated the role of transcriptional activation of the p53 downstream target, p21Waf1, on RPA2 phosphorylation, the stability of the p53/RPA complex and HR in cells undergoing replication arrest induced by camptothecin (CPT). We show that in CPT-treated cells, activation of p53 and p21Waf1 impedes RPA2 phosphorylation, while their depletion by siRNA stimulates it. The p53/RPA complex is more stable in wild-type cells than in cells depleted of p21Waf1. We used nocodazole-synchronized cells treated with CPT at the entrance to S phase to assess rates of HR. Regardless of their p53 or p21Waf1 status, the cells proceed through S phase at a similar rate and enter G2. While HR is low in wild-type cells and high in p53-depleted cells, only partial inhibition of HR is observed in the p21Waf1-depleted cells. This correlates with the extent of RPA sequestration by p53. Thus, in CPT-treated cells, p53-induced transcriptional activation of p21Waf1 regulates RPA2 phosphorylation, the stability of the p53/RPA complex and HR.

The Energetics and Physiological Impact of Cohesin Extrusion.

Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.

ATP-degrading ENPP1 is required for survival (or persistence) of long-lived plasma cells.

Survival of antibody-secreting plasma cells (PCs) is vital for sustained antibody production. However, it remains poorly understood how long-lived PCs (LLPCs) are generated and maintained. Here we report that ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is preferentially upregulated in bone marrow LLPCs compared with their splenic short-lived counterparts (SLPCs). We studied ENPP1-deficient mice (Enpp1 -/- ) to determine how the enzyme affects PC biology. Although Enpp1 -/- mice generated normal levels of germinal center B cells and plasmablasts in periphery, they produced significantly reduced numbers of LLPCs following immunization with T-dependent antigens or infection with plasmodium C. chabaudi. Bone marrow chimeric mice showed B cell intrinsic effect of ENPP1 selectively on generation of bone marrow as well as splenic LLPCs. Moreover, Enpp1 -/- PCs took up less glucose and had lower levels of glycolysis than those of wild-type controls. Thus, ENPP1 deficiency confers an energetic disadvantage to PCs for long-term survival and antibody production.

Associations of Autoimmunity, Immunodeficiency, Lymphomagenesis, and Gut Microbiota in Mice with Knockins for a Pathogenic Autoantibody.

A number of mouse strains transgenic for B-cell receptors specific for nucleic acids or other autoantigens have been generated to understand how autoreactive B cells are regulated in normal and autoimmune mice. Previous studies of nonautoimmune C57BL/6 mice heterozygous for both the IgH and IgL knockins of the polyreactive autoantibody, 564, produced high levels of autoantibodies in a largely Toll-like receptor 7-dependent manner. Herein, we describe studies of mice homozygous for the knockins that also expressed high levels of autoantibodies but, unlike the heterozygotes, exhibited a high incidence of mature B-cell lymphomas and enhanced susceptibility to bacterial infections. Microarray analyses and serological studies suggested that lymphomagenesis might be related to chronic B-cell activation promoted by IL-21. Strikingly, mice treated continuously with antibiotic-supplemented water did not develop lymphomas or abscesses and exhibited less autoimmunity. This mouse model may help us understand the reasons for enhanced susceptibility to lymphoma development exhibited by humans with a variety of autoimmune diseases, such as Sjögren syndrome, systemic lupus erythematosus, and highly active rheumatoid arthritis.

DNase-active TREX1 frame-shift mutants induce serologic autoimmunity in mice.

TREX1/DNASE III, the most abundant 3'-5' DNA exonuclease in mammalian cells, is tail-anchored on the endoplasmic reticulum (ER). Mutations at the N-terminus affecting TREX1 DNase activity are associated with autoimmune and inflammatory conditions such as Aicardi-Goutières syndrome (AGS). Mutations in the C-terminus of TREX1 cause loss of localization to the ER and dysregulation of oligosaccharyltransferase (OST) activity, and are associated with retinal vasculopathy with cerebral leukodystrophy (RVCL) and in some cases with systemic lupus erythematosus (SLE). Here we investigate mice with conditional expression of the most common RVCL mutation, V235fs, and another mouse expressing a conditional C-terminal mutation, D272fs, associated with a case of human SLE. Mice homozygous for either mutant allele express the encoded human TREX1 truncations without endogenous mouse TREX1, and both remain DNase active in tissues. The two mouse strains are similar phenotypically without major signs of retinal, cerebral or renal disease but exhibit striking elevations of autoantibodies in the serum. The broad range of autoantibodies is primarily against non-nuclear antigens, in sharp contrast to the predominantly DNA-related autoantibodies produced by a TREX1-D18N mouse that specifically lacks DNase activity. We also found that treatment with an OST inhibitor, aclacinomycin, rapidly suppressed autoantibody production in the TREX1 frame-shift mutant mice. Together, our study presents two new mouse models based on TREX1 frame-shift mutations with a unique set of serologic autoimmune-like phenotypes.

EBI2 overexpression in mice leads to B1 B-cell expansion and chronic lymphocytic leukemia-like B-cell malignancies.

Human and mouse chronic lymphocytic leukemia (CLL) develops from CD5+ B cells that in mice and macaques are known to define the distinct B1a B-cell lineage. B1a cells are characterized by lack of germinal center (GC) development, and the B1a cell population is increased in mice with reduced GC formation. As a major mediator of follicular B-cell migration, the G protein-coupled receptor Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) directs B-cell migration in the lymphoid follicles in response to its endogenous ligands, oxysterols. Thus, upregulation of EBI2 drives the B cells toward the extrafollicular area, whereas downregulation is essential for GC formation. We therefore speculated whether increased expression of EBI2 would lead to an expanded B1 cell subset and, ultimately, progression to CLL. Here, we demonstrate that B-cell-targeted expression of human EBI2 (hEBI2) in mice reduces GC-dependent immune responses, reduces total immunoglobulin M (IgM) and IgG levels, and leads to increased proliferation and upregulation of cellular oncogenes. Furthermore, hEBI2 overexpression leads to an abnormally expanded CD5+ B1a B-cell subset (present as early as 4 days after birth), late-onset lymphoid cancer development, and premature death. These findings are highly similar to those observed in CLL patients and identify EBI2 as a promoter of B-cell malignancies.

IL-21-driven neoplasms in SJL mice mimic some key features of human angioimmunoblastic T-cell lymphoma.

SJL/J mice exhibit a high incidence of mature B-cell lymphomas that require CD4(+) T cells for their development. We found that their spleens and lymph nodes contained increased numbers of germinal centers and T follicular helper (TFH) cells. Microarray analyses revealed high levels of transcripts encoding IL-21 associated with high levels of serum IL-21. We developed IL-21 receptor (IL21R)-deficient Swiss Jim Lambart (SJL) mice to determine the role of IL-21 in disease. These mice had reduced numbers of TFH cells, lower serum levels of IL-21, and few germinal center B cells, and they did not develop B-cell tumors, suggesting IL-21-dependent B-cell lymphomagenesis. We also noted a series of features common to SJL disease and human angioimmunoblastic T-cell lymphoma (AITL), a malignancy of TFH cells. Gene expression analyses of AITL showed that essentially all cases expressed elevated levels of transcripts for IL21, IL21R, and a series of genes associated with TFH cell development and function. These results identify a mouse model with features of AITL and suggest that patients with the disease might benefit from therapeutic interventions that interrupt IL-21 signaling.

Interactome maps of mouse gene regulatory domains reveal basic principles of transcriptional regulation.

A key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes marked alterations during ontogeny. However, the nature and extent of these changes are unclear. As part of the NIH Mouse Regulome Project, we here combined DNaseI hypersensitivity, ChIP-seq, and ChIA-PET technologies to map the promoter-enhancer interactomes of pluripotent ES cells and differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature extends to broadly transcribed genes, including Myc and Pim1 cell-cycle regulators, which associate with an entirely different set of enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting, we show that these enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during development correlates with promoter activity. We propose that organisms rely on a dynamic enhancer landscape to control basic cellular functions in a tissue-specific manner.

Homeostatic defects in B cells deficient in the E3 ubiquitin ligase ARF-BP1 are restored by enhanced expression of MYC.

The E3 ligase ARF-BP1 governs the balance of life and death decisions by directing the degradation of p53 and enhancing the transcriptional activity of MYC. We find B cells selectively deficient in ARF-BP1 have many defects in developing and mature B cells associated with increased expression of p53 and reduced expression of Myc. Overexpression of Myc results in suppression of p53 and complete reversal of defects induced by ARF-BP1 deficiency. These findings indicate that the dynamic balance between MYC and p53 required for normal B cell maturation and function is finely tuned and critically dependent on the activities of ARF-BP1.

Expression of plasma cell alloantigen 1 defines layered development of B-1a B-cell subsets with distinct innate-like functions.

Innate-like B-1a cells contribute significantly to circulating natural antibodies and mucosal immunity as well as to immunoregulation. Here we show that these classic functions of B-1a cells segregate between two unique subsets defined by expression of plasma cell alloantigen 1 (PC1), also known as ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1). These subsets, designated B-1a.PC1(lo) and B-1a.PC1(hi), differ significantly in IgH chain utilization. Adoptively transferred PC1(lo) cells secreted significantly more circulating natural IgM and intestinal IgA than PC1(hi) cells. In contrast, PC1(hi) cells produced more IL-10 than PC1(lo) cells when stimulated with LPS and phorbol 12-myristate 13-acetate (PMA). PC1(hi) cells were also more efficient than PC1(lo) cells in regulating Th1 cell differentiation, even though both B-1a subsets were comparably active in stimulating T-cell proliferation. Furthermore, PC1(lo) cells generated antigen-specific IgM responses to pneumococcal polysaccharide antigens, whereas PC1(hi) cells do not. We found that PC1(lo) cells develop from an early wave of B-1a progenitors in fetal life, whereas PC1(hi) cells are generated from a later wave after birth. We conclude that identification of B-1a.PC1(lo) and B-1a.PC1(hi) cells extends the concept of a layered immune system with important implications for developing effective vaccines and promoting the generation of immunoregulatory B cells.